During the current funding period, we have characterized several novel signaling pathways involving heterotrimeric G proteins and their binding proteins which are expressed in various endocrine cell lines including AtT-20 and GH3 pituitary cells, PC-12adrenal cells, and rat pituitary in situ. Our major accomplishments were: 1) Demonstration that the Golgi pool of calnuc resides in the Golgi for >24 hr, is secreted by the constitutive-like pathway and serves as a marker for this pathway; 2) Demonstration that the cytosolic pool of calnuc binds Ga subunits, redistributes Gai subunits and inhibits secretion from AtT-20 cells; 3) demonstration of a novel mechanism for regulation of Ga signaling through spatial segregation of GAIP, a GAP for Gai3, whereby GAIP meets Gai3 in clathrin-coated microdomains of the PM after agonist stimulation; 4) discovery of a new protein, GIPN, that serves as an E3 ligase promoting down-regulation of Gai subunits; 5) discovery of a new RGS family member that serves as a GAP for Gas and functions in sorting of growth factor receptors; 6) characterization of a PDZ protein, GIPC, that binds, GAIP, the TrkA growth factor receptor and APPL and attenuates MAP kinase activation; 7) Identification of a novel Ga binding protein, GIV, found in the Golgi and on COPI transport vesicles in the secretory pathway. Our overall goal for the coming renewal period is to further characterize the new pathways and to define the role of several of these proteins in regulated secretion and in endocytosis of G protein coupled receptors (GPCR) in order to extend knowledge of G protein mediated signaling pathways in endocrine and other cells. We will focus our attention on four specific aims: 1) To investigate whether calnuc, an EF hand, Ca2+ binding protein is essential for Ca2+-dependent, prohormone processing in AtT-20 cells and to identify the mechanism by which calnuc is retained in the Golgi in AtT-20 cells; 2) To determine the function of GIV (G protein Interacting protein on Vesicles) in the secretory pathway; 3) To elucidate the function and interactions of Gai3 and GAIP in the Golgi by live cell imaging on AtT-20 cells; 4) To determine the role of GIPC and its binding partners in linking endocytosis and signaling by LH receptors and D2 dopamine receptors. These studies can be expected to considerably expand knowledge of the role of G proteins in regulated secretion of hormones and in endocytosis and signaling of hormones by GPCR.